KR102350228B1 - Urinary exosome-derived biomarkers for diagnosis or prognosis of T cell-mediated rejection in kidney allografts - Google Patents

Abstract

The present invention relates to a biomarker for diagnosing non-invasive kidney transplant rejection and its use, and specifically, any one or more proteins selected from the group consisting of MGAM, SLC6A19, ACY3 and SLC5A10 or a gene encoding the same. Biomarker compositions and kits for diagnosing rejection or predicting the prognosis of kidney transplant rejection, a method of providing information necessary for diagnosing kidney transplant rejection or predicting the prognosis using the marker composition, providing information necessary for determining a treatment policy for kidney transplant rejection It relates to a method and a screening method for a therapeutic agent for rejection of a kidney transplant.

Description

Urinary exosome-derived biomarkers for diagnosis or prognosis of T cell-mediated rejection in kidney allografts

The present invention relates to a biomarker related to T cell-mediated rejection (TCMR) after kidney transplantation, a composition and kit for diagnosing or predicting T cell-mediated rejection, and a T cell-mediated rejection response using the same. It relates to a method of providing information for diagnosis or prediction.

The organ transplant field is slowly becoming a part of precision medicine. Acute rejection still occurs in 15 to 20% of kidney transplant recipients when using current standard immunosuppressive therapy, and to date, an increase in serum creatine levels or the development of new proteinuria is diagnosed by tissue biopsy. . Since the kidney status at the time of increased serum creatine concentration or new expression of proteinuria is already quite advanced in inflammation, there is a need for a biomarker capable of early detection of subclinical acute rejection. It is known that innate/acquired immune responses appear first at the molecular level before appearing at the histological level. Therefore, the development of non-invasive biomarkers for diagnosing and monitoring clinical status in advance after kidney transplantation is an essential step for precision medicine.

In the past, rejection after kidney transplantation was classified into hyperacute, acute, and chronic rejection according to the time of occurrence of rejection. Prior to Banff classification, the pathological diagnosis of transplanted kidneys was very simple. In particular, acute and chronic rejection were mainly classified according to the location of inflammatory cell infiltration. vascular) was classified as rejection. That is, from the current point of view, all of these are diagnostic classifications of T cell-mediated rejection except for hyperacute rejection. Currently, hyperacute rejection is understood as the most severe form of acute ABMR.

Regarding the diagnosis of rejection after kidney transplantation, urine biomarkers for diagnosing acute rejection after kidney transplantation are known (Suthanthiran et al., Urinary-Cell mRNA Profile and Acute Cellular Rejection in Kidney Allografts. The New England Journal of Medicine (2013) 369;1). However, we discovered a biomarker that can distinguish T cell-mediated rejection from antibody-mediated rejection, and furthermore, a biomarker that can differentiate and diagnose BK virus nephropathy and transplant rejection, which is a complication after kidney transplantation. There are no studies that are intended to be used to select a treatment direction after transplantation and to judge the course of treatment.

As a result of earnest efforts to discover non-invasive and accurate kidney transplant rejection diagnostic markers, the present inventors identified new biomarkers that can predict the presence or absence of kidney transplant rejection and types of rejection through a proteomics approach. invention was completed.

In the present invention, kidney transplant rejection diagnosis or prognosis in urine exosomes of normal control group (NOMOA), antibody-mediated rejection group (ABMR), T cell-mediated rejection group (TCMR) and BK virus infectious nephropathy group (BKVN) By discovering predictive markers, we intend to contribute to judging the course of treatment after kidney transplantation and selecting a treatment method.

In one aspect, the present invention provides a biomarker composition for diagnosing kidney transplant rejection or predicting prognosis, comprising any one or more proteins selected from the group consisting of MGAM, SLC6A19, ACY3 and SLC5A10 or genes encoding them.

In another aspect, the present invention provides a diagnosis of rejection after kidney transplantation comprising a material for measuring the mRNA expression level of any one or more proteins selected from the group consisting of MGAM, SLC6A19, ACY3 and SLC5A10 or a gene encoding the protein. A composition for prognosis prediction is provided.

In another aspect, the present invention provides a kit for diagnosing rejection or predicting prognosis after kidney transplantation comprising the composition.

In another aspect, the present invention comprises measuring the mRNA expression level of any one or more proteins selected from the group consisting of MGAM, SLC6A19, ACY3, and SLC5A10 in a sample obtained from an individual or a gene encoding the same. It provides a method to provide information necessary for the diagnosis of rejection.

In another aspect, the present invention comprises measuring the mRNA expression level of any one or more proteins selected from the group consisting of MGAM, SLC6A19, ACY3, and SLC5A10 in a sample isolated from an individual or a gene encoding the same. Kidney It provides a method for providing information necessary for decision-making in the treatment of transplant rejection.

In another aspect, the present invention relates to any one or more proteins selected from the group consisting of MGAM, SLC6A19, ACY3 and SLC5A10 in urine or a gene encoding the same after administration of a drug candidate for treatment of kidney transplant rejection to mammals other than humans It provides a screening method for a therapeutic agent for rejection of kidney transplant, comprising the step of measuring the expression level of mRNA.

Hereinafter, the present invention will be described in more detail.

In one embodiment of the present invention, for the discovery of specific exosome proteins in urine samples of kidney transplant patients and donors, candidate proteins were selected based on proteomics analysis. A total of 1820 proteins were detected in urine exosome protein quantitative analysis, and 13 proteins with significantly different expression profiles between the "NOMOA and DONOR group" and "TCMR group" were selected. Finally, four proteins showing significantly different expression patterns from those of the ABMR group or the BKVN group were identified among the 13 proteins. The identified proteins are MGAM (Maltase-glucoamylase, intestinal; UniProt Accession No. O43451), SLC6A19 (Sodium-dependent neutral amino acid transporter B(0)AT1, sodium-dependent neutral amino acid transporter B(0) AT1, UniProt Accession No. Q695T7), ACY3 (N-acyl-aromatic-L-amino acid amidohydrolase, N-acyl-aromatic-L-amino acid amidohydrolase, UniProt Accession No. Q96HD9), SLC5A10 (Sodium/glucose) cotransporter 5, sodium/glucose cotransporter 5, UniProt Accession No. A0PJK1).

Therefore, in the present invention, selected biomarkers involved in poor prognosis after kidney transplantation, that is, MGAM (Maltase-glucoamylase), SLC6A19 (Sodium-dependent neutral amino acid transporter B(0)AT1), ACY3 (N-acyl-aromatic) -L-amino acid amidohydrolase), SLC5A10 (Sodium/glucose cotransporter 5) containing any one or more proteins selected from the group consisting of or a gene encoding the same, providing a biomarker composition for diagnosing kidney transplant rejection or predicting prognosis do. Using this, it is possible to predict the patient's prognosis after kidney transplantation, and to determine an appropriate treatment direction according to the predicted prognosis, so that it is possible to provide a customized treatment method and reduce the mortality rate of kidney transplant patients with poor prognosis.

As used herein, the term "renal allograft rejection" refers to a series of immune responses that occur when the recipient's immune system recognizes the transplanted kidney as a foreign antigen. Depending on the findings, antibody-mediated rejection (ABMR), T cell mediated rejection (TCMR), and all of these can be classified into mixed rejection. have.

In the present invention, the rejection of the kidney transplant may include all of antibody-mediated rejection, T-cell-mediated rejection, and mixed rejection, and preferably, acute T-cell mediated rejection (TCMR). ), acute antibody-mediated rejection (CAMR), chronic active antibody-mediated rejection, and mixed rejection.

As used herein, the term "biomarker" refers to a molecule quantitatively or qualitatively related to the existence of a biological phenomenon, and the marker of the present invention predicts a gene that can confirm whether there is rejection after kidney transplantation or a patient with a good or poor prognosis. Refers to the gene that is the basis for A marker may be derived from a genomic nucleotide sequence or from an expressed nucleotide sequence (eg, from RNA, nRNA, mRNA, cDNA, etc.), or from an encoded polypeptide. The term includes nucleic acids used as pairs of probes or primers capable of amplifying a nucleic acid sequence complementary to or flanking a marker sequence, such as a marker sequence.

According to an embodiment of the present invention, among kidney transplant patients, the urine of a patient exhibiting T-cell-mediated rejection (TCMR) compared to a normal control (no major abnormality, NOMOA) without major abnormalities on histological examination By confirming that the biomarkers were specifically expressed, it was found that the biomarkers can be utilized as diagnostic markers of T cell-mediated rejection.

In the present invention, the 'expression' means that a protein or nucleic acid is produced in a cell. 'Protein' is used interchangeably with 'polypeptide' or 'peptide' and refers to, for example, a polymer of amino acid residues as commonly found in proteins in their natural state. 'Polynucleotide' or 'nucleic acid' refers to deoxyribonucleotides (DNA) or ribonucleotides (RNA) in single- or double-stranded form. Unless otherwise limited, known analogs of natural nucleotides that hybridize to nucleic acids in a manner analogous to naturally occurring nucleotides are also included. 'mRNA' refers to RNA that transfers genetic information (gene-specific base sequence) to the ribosome, which specifies the amino acid sequence from a specific gene during protein synthesis.

In the present invention, 'diagnosis' means confirming the presence or characteristics of a pathological condition. Diagnosis in the present invention is to measure the level of any one or more proteins or mRNAs selected from the group consisting of MGAM, SLC6A19, ACY3, and SLC5A10 to confirm the presence or occurrence of kidney transplant rejection pathology.

In the present invention, "prediction of prognosis" means presuming the medical attribution, and for the purpose of the present invention, the course of the disease of the patient who received a kidney transplant (disease progression, improvement, transplant rejection reaction, drug resistance) means to guess in advance.

The present invention also provides a composition for diagnosing or predicting the prognosis of rejection after kidney transplantation, comprising a substance for measuring the expression level of mRNA or protein thereof of the biomarker of the present invention.

When the composition for diagnosis or prognosis of the present invention is for measuring the mRNA expression level, the material for measuring the mRNA expression level is MGAM, SLC6A19, ACY3 and SLC5A10 Any one or more genes or mRNA specific It may be a probe or primer set that binds to

In the present invention, a "primer" is a nucleic acid sequence having a short free 3' hydroxyl group, which can form a base pair with a complementary template, and serves as a starting point for copying the template. It refers to a short nucleic acid sequence that functions. In the present invention, PCR amplification is performed using the sense and antisense primers of the marker polynucleotide of the present invention, and the rejection of kidney transplantation can be diagnosed or the prognosis can be predicted based on whether a desired product is produced. PCR conditions, the length of the sense and antisense primers can be modified based on what is known in the art.

As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to several bases to several hundred bases in length that can form specific binding to mRNA, and is labeled The presence or absence of specific mRNA can be checked. The probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like. In the present invention, hybridization is carried out using a probe complementary to the marker polynucleotide of the present invention, and the diagnosis or prognosis of kidney transplant rejection can be predicted through hybridization. Selection of appropriate probes and hybridization conditions can be modified based on those known in the art.

The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method or other well-known methods. Such nucleic acid sequences may also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, encapsulation, substitution of one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phosphotriesters, phosphoros. amidates, carbamates, etc.) or charged linkages (eg phosphorothioates, phosphorodithioates, etc.).

Meanwhile, in the present invention, the substance for measuring the protein expression level of the biomarker may be an antibody that specifically binds to any one or more proteins selected from the group consisting of MGAM, SLC6A19, ACY3 and SLC5A10.

The "antibody" is a term known in the art and refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker of the present invention, and such an antibody is obtained by cloning each gene into an expression vector according to a conventional method to obtain a protein encoded by the marker gene , can be prepared by a conventional method from the obtained protein. This also includes a partial peptide that can be made from the protein, and the partial peptide of the present invention contains at least 7 amino acids, preferably 9 amino acids, more preferably 12 or more amino acids. The form of the antibody of the present invention is not particularly limited, and any part thereof is included in the antibody of the present invention as long as it has polyclonal antibody, monoclonal antibody, or antigen-binding property, and all immunoglobulin antibodies are included. Furthermore, the antibody of the present invention includes a special antibody such as a humanized antibody. The antibody against the protein encoded by the biomarker gene of the present invention may be any antibody that can be prepared by methods known in the art. For example, the antibody used for the detection of the kidney transplant rejection diagnostic or prognostic marker of the present invention includes a complete form having two full-length light chains and two full-length heavy chains, as well as functional fragments of antibody molecules. can do. The functional fragment of the antibody molecule refers to a fragment having at least an antigen-binding function, and may be Fab, F(ab'), F(ab')2, Fv, etc., but is not particularly limited thereto.

In another aspect, the present invention provides a kit for diagnosing kidney transplant rejection or predicting the prognosis, comprising the composition for predicting or diagnosing kidney transplant rejection.

The kit of the present invention may include one or more other component compositions, solutions, or devices suitable for the analysis method, as well as primers and probes that recognize the antibody or mRNA that recognizes the eight proteins as markers.

In a specific embodiment, the kit may be a diagnostic kit comprising essential elements necessary for performing a reverse transcription polymerase reaction. The reverse transcription polymerase reaction kit includes each primer pair specific for a marker gene. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of each marker gene, and has a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. It may also include a primer specific for the nucleic acid sequence of the control gene. Other reverse transcription polymerase reaction kits include test tubes or other suitable containers, reaction buffers (with varying pH and magnesium concentrations), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC -Water (DEPC-water), sterile water, etc. may be included.

In another aspect, it may be a diagnostic kit comprising essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate to which cDNA or oligonucleotide corresponding to a gene or fragment thereof is attached, and reagents, agents, enzymes, etc. for preparing a fluorescently labeled probe. The substrate may also contain cDNA or oligonucleotides corresponding to control genes or fragments thereof.

In another aspect, the present invention comprises measuring the mRNA expression level of any one or more proteins selected from the group consisting of MGAM, SLC6A19, ACY3 and SLC5A10 or a gene encoding the same in a sample obtained from an individual. Kidney transplantation, comprising the steps of: It provides a method to provide information necessary for the diagnosis of rejection.

In the present invention, the "individual" may include, without limitation, mammals, farmed fish, etc., including rats, livestock, humans, etc. that are likely to or have developed kidney transplant rejection.

In the present invention, the "sample" used for analysis is blood, plasma, serum, saliva, nasal fluid, sputum, ascites, vaginal secretion, urine, feces, etc. Kidney transplant rejection specific protein that can be distinguished from normal conditions to be identified. including biological samples that can be Preferably, it may be a biological liquid sample, such as blood, serum, plasma or urine, and most preferably urine. The sample may be prepared to increase the detection sensitivity of the protein marker. For example, a sample obtained from a patient may be subjected to anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, continuous It may be pre-treated using a method such as sequential extraction or gel electrophoresis.

In the present invention, the "measurement of the expression level of a protein" is a process of confirming the presence and level of expression of a protein expressed in a marker gene in a biological sample in order to diagnose rejection of a kidney transplant or predict a prognosis. The presence or absence of a protein can be detected or the amount of a protein can be measured using an antibody that specifically binds. Antibodies specific to the protein are as described in the composition for diagnosis or prognosis of the present invention. Methods for measuring the expression level of proteins can be used without limitation, methods known in the art, for example, western blotting (western blotting), dot blotting (dot blotting), enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay) , ELISA), radioimmunoassay (RIA), radioimmunodiffusion, ochteroni immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation, complement fixation assay, flow cytometry (FACS) or protein There is a chip method and the like, but is not limited thereto.

In the present invention, the "measuring the expression level of mRNA" is a process of confirming the presence and expression level of mRNA of a marker gene in a biological sample in order to diagnose rejection of kidney transplantation or predict the prognosis, by measuring the amount of mRNA Able to know. Analysis methods for this include RT-PCR, competitive RT-PCR (RT-PCR), real-time RTPCR (RT-PCR), RNase protection method, northern blotting, or DNA There is a chip (DNA chip technology), but is not limited thereto.

In the method of the present invention, when the result of comparing the measured protein or mRNA expression level with the protein or mRNA expression level of a normal control sample corresponds to any one of a) to d) below, it is determined as a T cell-mediated rejection It may further include the step of

a) upregulation of MGAM;

b) upregulation of SLC6A19;

c) upregulation of ACY3;

d) upregulation of SLC5A10;

The normal control group is a normal person who has not received a kidney transplant or a kidney transplant patient who has undergone a kidney transplant, but exhibits stable kidney function and does not exhibit antibody-mediated rejection, T-cell-mediated rejection, or mixed rejection. It is possible to accurately predict the prognosis of a patient whose prognosis is to be known by obtaining and comparing the gene expression level or expression pattern from a sample collected from a kidney transplant and a sample collected from a patient who wants to know the presence or prognosis of kidney transplant rejection.

Therefore, according to the present invention, it is possible to accurately diagnose or predict rejection and its prognosis after kidney transplantation, and to establish an appropriate treatment plan according to the diagnosed or predicted prognosis can be obtained.

Accordingly, the present invention also includes measuring the mRNA expression level of any one or more proteins selected from the group consisting of MGAM, SLC6A19, ACY3, and SLC5A10 in a sample isolated from an individual or a gene encoding the same. It provides a method to provide information necessary for decision-making on the treatment policy for rejection.

In another aspect, the present invention relates to any one or more proteins selected from the group consisting of MGAM, SLC6A19, ACY3, and SLC5A10 in urine or a gene encoding the same after administration of a candidate drug for a treatment for kidney transplant rejection to a mammal other than a human. It provides a screening method for a therapeutic agent for rejection of kidney transplant, comprising the step of measuring the expression level of mRNA.

Specifically, it is a method of comparing the increase or decrease in mRNA or protein expression of a marker in the presence and absence of a kidney transplant rejection treatment candidate, which can be usefully used for screening kidney transplant rejection therapeutic agents. Substances that indirectly or directly increase or decrease the expression level of mRNA or protein of a marker can be selected as a therapeutic agent for kidney transplant rejection.

That is, the expression level of the marker of the present invention is measured in a biological sample obtained from a mammal other than a human in the absence of a candidate substance for treatment of kidney transplant rejection, and the expression level of the marker of the present invention is measured in the presence of a candidate for treatment of kidney transplant rejection. After comparing the two by measuring the expression level, a substance that increases or decreases the expression level of the marker of the present invention in the presence of a candidate substance for treatment of kidney transplant rejection may be selected as a therapeutic agent for kidney transplant rejection. there will be

Since the expression of the biomarker provided in the present invention is significantly increased in patients exhibiting renal transplant rejection, the diagnostic composition, kit or detection method of the present invention comprising a substance for measuring the expression level of the biomarker of the present invention Kidney transplant rejection can be accurately and quickly diagnosed.

In addition, since the biomarker of the present invention is specifically expressed in T-cell-mediated rejection patients differently from other types of transplant rejection or BK virus nephropathy, which is common after transplantation, it is possible to accurately determine whether a T-cell-mediated rejection reaction occurs. and can be identified quickly, and can provide the information necessary to make a subsequent treatment policy decision.

1 is a diagram showing the selection process of biomarkers representing the ABMR, TCMR and BKVN groups.
2 is a view showing the results of selecting a total of 63 proteins present at the intersection site as biomarker candidates for TCMR as a result of a t-test and a volcano plot.
3 shows the antibody-mediated rejection group (ABMR), T-cell-mediated rejection group (TCMR) and BK virus-infectious nephropathy group ( BKVN), respectively, 63, 108, and 53 urine exosomal proteins are shown as a Venn diagram showing the results of confirming that they are expressed significantly differently from other groups.
4 is a diagram showing the final selection results of four proteins having significantly different expression patterns compared to ABMR and BKVN group proteins among TCMR biomarker candidates.
5 is a result of performing ROC curve analysis by analyzing the expression levels of four T-cell-mediated rejection markers selected in Examples of the present invention.

The present invention will be described in more detail with reference to the following examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

<Experiment method>

Selection of patients and preparation of urine samples

In order to discover biomarkers for diagnosing T cell-mediated rejection in kidney transplant patients, a total of 60 patients including patients and donors who underwent an indication biopsy within 5 years of kidney transplantation were biopsied 2 Urine samples were collected ˜3 hours before. In addition, urine samples were collected from living kidney donors just before kidney donation surgery. Patients were divided into 5 groups according to the pathological diagnosis of biopsy; 12 antibody-mediated rejection (ABMR) groups, 8 T-cell mediated rejection (TCMR) groups, 5 BK viral nephropathy (BKVN) groups, 11 no major abnormalities (NOMOA) groups, and donors ( DONOR) 24 counties. Urine from patients with mixed allogeneic lesions was excluded. This study was conducted with the approval of the Asan Hospital Medical Institution Evaluation Committee, and written consent was obtained from all patients.

Processing of urine samples and isolation of exosomes

에서 15분 간 원심분리하였고, 엑소좀을 추출할 때까지 -80

에 보관하여 실험에 사용하였다.A previously reported Pisitkun et al. (2004) and Alvarez et al. (2012) was slightly modified to separate exosomes by a step-wise ultracentrifugation method. Specifically, more than 30 mL of urine samples were collected from each experiment participant, and in each urine sample, 400 μl protease inhibitor mixture [50 μM 4-(2-animoethyl) benzensulfonyl fluoride hydrolchloride (AEBSF-HCl, Sigma-Aldrich), 2 μM Leupeptin-hemisulfate (Sigma-Aldrich), and 3.3 mM Sodium azide (Sigma-Aldrich)] were added. To remove urinary sediments, including whole cells, large membrane particles, and other debris, urine samples were run at 4,000 rpm, 4

was centrifuged for 15 minutes at -80 until extraction of exosomes.

was stored and used for the experiment.

Thereafter, the frozen urine samples were thawed by 15 mL each, vortexed for 1 minute, centrifuged at 17,000xg for 15 minutes at room temperature, and the supernatant (SN1) was collected. SN1 was ultracentrifuged at 200,000 x g for 70 min at room temperature using a Beckman Coulter Optima L-80xp ultracentrifuge, rotor SW40Ti (Beckman Coulter, Brea, CA, USA). The supernatant was discarded, the pellet was washed by dissolving in 11 mL of DPBS, and then ultracentrifuged again at 200,000 x g for 70 min at room temperature. The supernatant was discarded and the exosome pellet was used for protein isolation or exosome particle quantification.

LC-MS analysis and protein identification

Sample preparation for proteomics analysis went through lyophilization, protein solubilization and digestion. The resulting peptide mixture was desalted, dried using C18 reverse phase chromatography and analyzed by high resolution mass spectrometry coupled with nano-flow liquid chromatography.

Sequence database analysis and label free quantitation (LFQ) analysis were applied using Proteome Discoverer 2.2 (Thermo Fisher Scientific) to search for biomarker candidates for diagnosing or predicting kidney transplant rejection. From this, 1820 urine exosome proteins were identified. Gene ontology assignments, molecular function and Kegg pathway analysis were performed using the DAVID bioinformation database.

Biomarker candidate selection

The process of selecting a protein as a biomarker candidate is summarized in FIG. 1 . To select specific biomarkers representative of each pathological group, t-tests were used to select proteins with significantly different mean abundances compared to the NOMOA and DONOR groups. Also, using a volcano plot, proteins showing a difference of more than 2 fold compared to the NOMOA group were selected. In the two analyzes, overlapping proteins were selected and compared again with the other two pathological groups to compare significant differences in mean abundance. All analyzes were performed with a cut off of p <0.05.

<Experiment result>

1. Identification and primary screening of protein biomarkers in TCMR patients

To select specific biomarkers representing each pathological group from 4193 urine proteins, sequence database analysis and label free quantitation (LFQ) analysis were applied using Proteome Discoverer 2.2 (Thermo Fisher Scientific). 1820 urine exosome proteins were identified.

In order to select a marker capable of distinguishing the TCMR group from among 1820 urine exosome proteins, first, a protein having a significantly different average abundance in the TCMR group compared with the NOMOA and Donor groups was first tested using the T test. As a result of selection, it was confirmed that 63 proteins were specifically expressed only in the TCMR group (p<0.05).

Next, by comparing only the NOMOA group and the TCMR group, proteins showing a difference in expression level of more than two-fold were identified. As a result, 730 proteins were selected (p<0.05).

Among the 730 proteins showing a 2-fold or more difference in expression level with the 63 proteins selected above, 13 overlapping proteins were finally selected as TCMR marker candidates, and the significant difference in abundance with the ABMR group and BKVN was compared again. The results are shown in [Table 1] below.

[Table 1]

List of proteins showing differences in NOMOA, DONOR and TCMR groups (13 types)

2. Final selection of TCMR-specific biomarkers

Final candidate proteins were selected based on T-test analysis to discover specific exosome proteins in the ABMR, TCMR and BKVN groups, respectively. Among the proteins specifically expressed only in the TCMR group compared to the NOMOA group or the DONOR group among the urine exosome proteins, only those proteins whose expression profile differs by more than two-fold compared to the NOMOA group are selected through the process of selecting those of [Table 1]. Thirteen types of proteins were first selected.

Among the 13 primary proteins selected in [Table 1], a protein having a significantly different average abundance only in the TCMR group compared to the ABMR group was selected as a candidate biomarker, and compared to the BKVN group, only in the TCMR group. Proteins with significantly different average abundances were selected as candidate biomarkers, and finally, four overlapping proteins among the candidate biomarker proteins were selected as final TCMR biomarker proteins, which are expected to differ in ABMR, BKVN, and TCMR groups. was selected as The identified proteins are Maltase-glucoamylase, intestinal (alias: MGAM, UniProt O43451), sodium-dependent neutral amino acid transporter B(0)AT1 (Sodium-dependent neutral amino acid transporter B(0)AT1, alias: MGAM, UniProt O43451) : SLC6A19, UniProt Q695T7), N-acyl-aromatic-L-amino acid amidohydrolase (N-acyl-aromatic-L-amino acid amidohydrolase, alias: ACY3, UniProt Q96HD9), sodium/glucose cotransporter 5 ( Sodium/glucose cotransporter 5, alias: SLC5A10, UniProt A0PJK1).

3. Confirmation of characteristics of selected TCMR-specific biomarkers

In order to confirm the characteristics of the finally selected biomarkers, gene ontology assignments, molecular functions, and Kegg pathway analysis were performed using the DAVID bioinformation database, and the results are shown in [Table 2].

[Table 2]

Functional Characterization of Selected TCMR-Specific Markers

As shown in [Table 2], it was confirmed that most of the selected TCMR-specific markers were not sufficiently known for their functional properties.

4. Validation of TCMR-specific biomarkers

For the detection of specific exosomal proteins in urine samples from 46 kidney transplant patients and donors, candidate proteins were selected based on proteomics analysis. A total of 1820 proteins were detected in urine exosome protein quantitative analysis. Among them, 13 proteins with significantly different expression profiles between the "NOMOA and DONOR group" and the "TCMR group" were selected. Finally, among the 13 proteins, four proteins (MGAM, SLC6A19, ACY3, and SLC5A10) showing significantly different expression patterns from those of the ABMR group or BKVN group were identified and selected as final TCMR-specific markers.

The potential of the biomarkers to distinguish the NOMOA, ABMR, BKVN group and the TCMR group through ROC analysis was evaluated and shown in FIG. 5 . As a result, it was confirmed that the area under the curve was 0.5 or more for all biomarkers, indicating a very high level in sensitivity and specificity.

Claims (11)

MGAM (Maltase-glucoamylase), ACY3 (N-acyl-aromatic-L-amino acid amidohydrolase) and SLC5A10 (Sodium / glucose cotransporter 5) comprising any one or more proteins selected from the group consisting of or a gene encoding the same, kidney A biomarker composition for diagnosing or predicting prognosis of T cell-mediated rejection after transplantation.
delete Any one or more proteins selected from the group consisting of MGAM (Maltase-glucoamylase), ACY3 (N-acyl-aromatic-L-amino acid amidohydrolase) and SLC5A10 (Sodium/glucose cotransporter 5) or mRNA of a gene encoding the protein A composition for diagnosing or predicting prognosis of T-cell-mediated rejection after kidney transplantation, comprising a substance for measuring the expression level.
4. The method of claim 3,
The composition for measuring the expression level of the mRNA is a primer or probe that specifically binds to the gene.
4. The method of claim 3,
The substance for measuring the expression level of the protein is an antibody, an interacting protein, a ligand, an oligopeptide, a peptide nucleic acid (PNA), a nanoparticle or an aptamer that specifically binds to the protein or a fragment thereof.
A kit for diagnosing or predicting prognosis of T cell-mediated rejection after kidney transplantation, comprising the composition of claim 3.
MGAM (Maltase-glucoamylase), SLC6A19 (Sodium-dependent neutral amino acid transporter B(0)AT1), ACY3 (N-acyl-aromatic-L-amino acid amidohydrolase) and SLC5A10 (Sodium/ Including the step of measuring the mRNA expression level of any one or more proteins selected from the group consisting of glucose cotransporter 5) or a gene encoding the same,
If the result of comparing the measured protein or mRNA expression level with the protein or mRNA expression level of the normal control sample corresponds to any one or more of the following a) to d), determining a T cell-mediated rejection after kidney transplantation A method of providing information necessary for diagnosing T cell-mediated rejection after kidney transplantation, which further comprises:
a) upregulation of MGAM;
b) upregulation of SLC6A19;
c) upregulation of ACY3; and
d) Upregulation of SLC5A10.
delete delete In urine-derived exosomes isolated from individuals, MGAM (Maltase-glucoamylase), SLC6A19 (Sodium-dependent neutral amino acid transporter B(0)AT1), ACY3 (N-acyl-aromatic-L-amino acid amidohydrolase) and SLC5A10 (Sodium) /glucose cotransporter 5) comprising the step of measuring the expression level of the mRNA of any one or more proteins selected from the group consisting of or a gene encoding the same,
If the result of comparing the measured protein or mRNA expression level with the protein or mRNA expression level of a normal control sample corresponds to any one or more of the following a) to d), determining a T cell-mediated rejection after kidney transplantation A method of providing information necessary for determining a treatment policy for T-cell-mediated rejection after kidney transplantation, which further comprises:
a) upregulation of MGAM;
b) upregulation of SLC6A19;
c) upregulation of ACY3; and
d) Upregulation of SLC5A10.
(i) MGAM (Maltase-glucoamylase) in urine-derived exosomes, SLC6A19 (Sodium-dependent neutral amino acid transporter B(0)) AT1), ACY3 (N-acyl-aromatic-L-amino acid amidohydrolase) and SLC5A10 (Sodium / glucose cotransporter 5) any one or more proteins selected from the group consisting of or measuring the mRNA expression level of a gene encoding the same ;
(ii) comparing the expression level measured in step (i) with the expression level before administration of the candidate substance; and
(iii) if the result compared in step (ii) corresponds to any one of a) to d) below, selecting the candidate material as a therapeutic agent for T-cell-mediated rejection after kidney transplantation; Screening method for therapeutic agents for T-cell-mediated rejection after transplantation:
a) upregulation of MGAM;
b) upregulation of SLC6A19;
c) upregulation of ACY3; and
d) Upregulation of SLC5A10.

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